Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens.

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14-16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

Multiplex comparison of the digestibility of allergenic and non-allergenic proteins in rice grains by in vitro digestion.

To evaluate the digestibility of rice allergenic and nonallergenic proteins under the influence of the rice grain matrix, rice powder was subjected to in vitro digestion by simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Rice proteins were extracted from the liquid and the solid phases and analysed by SDS-PAGE, and rice allergenic proteins were detected by a multiplex immunodetection method. The digestion of soluble proteins was carried out in both liquid and solid phases, while that of insoluble proteins only occurred in the solid phase. In SGF digestion, rice proteins were more quickly digested at pH 1.2 than at pH 2.0 or 2.5. Moreover, the digestibility of five kinds of rice allergenic proteins was influenced by pH level, heat processing, starch matrix, solubility, and protein properties, on a case-by-case basis. On the other hand, all detected rice allergenic proteins and non-allergenic proteins were rapidly digested in SIF.

Evaluation of extraction solutions for biochemical analyses of the proteins in rice grains.

The influence of different extraction solutions on the proteins extracted from rice grains was investigated. The largest amounts of salt-soluble proteins were extracted with solutions supplemented with Tris-HCl at pH 8.0. Rice allergens were analyzed by multiplex immunodetection. Except for α-globulin extracted with the solutions at pH 8.0, which showed a low-molecular-weight band besides the main band, no significant solution-dependent difference among the allergens was found. Total proteins were extracted with four kinds of solution. The extraction of the basic subunit of glutelin was found to be SDS-dependent, and more protein was obtained with extraction solutions supplemented with SDS. The contents of α-globulin and α-amylase/trypsin inhibitors were higher in the extracts without SDS than with SDS. We conclude from the present data that, in order to obtain comparable data from rice grain salt-soluble and total protein analyses, differences in the protein extraction efficiency of solutions used should be taken into consideration.

Mutational analysis of the inducer recognition sites of the LysR-type transcriptional regulator TfdT of Burkholderia sp. NK8.

TfdT is a LysR-type transcriptional regulator that activates the transcription of the chlorocatechol degradative gene operon tfdCDEF of the chlorobenzoate-degrading bacterium Burkholderia sp. NK8. To identify the amino acids involved in the effector recognition by TfdT, a polymerase-chain-reaction-based random mutagenesis protocol was applied to introduce mutations into the tfdT gene. Nine types of TfdT mutant bearing a single-amino-acid substitution at positions, Lys-129, Arg-199, Val-226, Val-246, and Pro-267 were obtained on the basis of their altered effector profiles and enhanced responses particularly to 2-chlorobenzoate, 2-aminobenzoate, and 2,6-dichlorobenzoate. All the TfdT mutants showed enhanced response to the effectors with a chloro-group in C-2 of benzoic acid. A homology model of wild-type TfdT was built on the basis of the crystal structure of CbnR with SwissModel. In this model, residues corresponding to the mutation sites of isolated TfdT mutants were located at the interface between the domains RD-I and RD-II. The findings that these TfdT mutants expressed altered effector specificities and enhanced responses to specific effectors suggest that these five residues are involved in effector binding by TfdT.

Two kinds of chlorocatechol 1,2-dioxygenase from 2,4-dichlorophenoxyacetate-degrading Sphingomonas sp. strain TFD44.

Two kinds of chlorocatechol 1,2-dioxygenase (CCD), TfdC and TfdC2 were detected in Sphingomonas sp. strain TFD44. These two CCDs could be simultaneously synthesized in TFD44 during its growth with 2,4-D as the sole carbon and energy sources. The apparent subunit molecular masses of TfdC and TfdC2 estimated by SDS-PAGE analysis were 33.8 and 33.1 kDa, respectively. The genes encoding the two CCDs were cloned and expressed in Escherichia coli. The two purified CCDs showed broad substrate specificities but had different specificity patterns. TfdC showed the highest specificity constant for 3-chlorocatechol and TfdC2 showed the highest specificity constant for 3,5-dichlorocatechol. The substrate specificity difference seemed to correlate with the alternation of amino acid supposed to be involved in the interaction with substrates. Whereas phylogenetic analysis indicated that the CCDs of Sphingomonas constitute a distinctive group among Gram-negative bacteria, TfdC and TfdC2 of TFD44 have divergently evolved in terms of their substrate specificity.

Detection of telomerase activity in tissues and primary cultured lymphoid cells of Penaeus japonicus.

Telomerase is a ribonucleoprotein enzyme that can elongate telomeric DNA, which is thought to be required for the development of cellular immortality and oncogenesis in mammals. We examined telomerase activity in tissues and primary cultured lymphoid cells of adult penaeid shrimps. Using the telomeric repeat amplification protocol (TRAP), we studied the characteristics of a putative novel telomerase in Penaeus japonicus. This telomerase could be inactivated by heating or treatment with RNase A or proteinase K. At elongation, this telomerase required dATP, dGTP, and dTTP, but not dCTP, as substrates. Sequence analysis of the TRAP product revealed that this telomerase synthesized (TTAGG)(n) repeated sequences. The activity of this telomerase was decreased but still readily detectable in 100 ng of protein extract from lymphoid tissue. The telomerase activity was detected in all examined tissues including testis, ovary, lymphoid, heart, hepatopancreas, and muscle. The highest telomerase activity was in the extract of ovarian tissues. In primary cultured lymphoid cells, the telomerase activity was retained. Thus, primary cultured lymphoid cells of Penaeus japonicus possess one of the factors necessary for cell line establishment.

Penaeid (Penaeus japonicus) lymphoid cells replicate by cell division in vitro.

Penaeid cell culture has gained much attention as a potential model to facilitate researches on the characterization of the virus and to develop more sophisticated and improved diagnostic procedures for use in the aquaculture industry. However, to date, cell division processes of cultured penaeid cells have not been found, which is suggested as one of the reasons that block the establishment of the continuous penaeid cell lines. We reported here the cell division processes of cultured lymphoid cells of Penaeus japonicus. The culture medium used was based on M199 and was modified by supplementing saline components. Cultures were incubated at 25 degrees C, and 5% CO2 was supplemented. In primary cultured lymphoid cells, dividing cells in different shapes were found. Cell division processes of 12 dividing lymphoid cells were tracked. After cell division, their daughter cells turned into fibroblast-like or epithelioid cells. These results proved that the culture conditions used were suitable for lymphoid cells of I japonicus to proliferate in vitro and that cultured lymphoid cells still had the ability to carry out cell division. These findings would give light to the establishment of continuous penaeid cell lines and would also provide us with the knowledge of cell division processes of the penaeid.